In view of the public outcry against the incineration of stockpiles of chemical agents, the U.S. Army is seeking alternative chemical agent demilitarization technologies. Microbial biodegradation is one promising alternative the Army is considering for this purpose. Recently, microbial biodegradation of hydrolyzed mustard (bis-2-chlorethyl sulfide) was sanctioned as the most promising alternative technology suitable for the Army's demilitarization goals for that chemical agent. See Irvine, D. A., J. P. Earley, D. P. Cassidy and S. P. Harvey, “Biodegradation of Sulfur Mustard Hydrolysate in the Sequencing Batch Reactor,” Water Sci. and Tech. 35: 67–74 (1996), incorporated herein by reference in its entirety.
As other chemical warfare agents have different structures, the biodegradation of these materials and/or their neutralization products were pursued. The organophosphorus nerve agents VX (O-ethyl-S-2(diisopropylaminoethyl)methylphosphonothioate) and Sarin (0 isopropylmethylphosphofluoridate) represent a large portion of the Army's stockpile. Equimolar mixtures of VX and water undergo a slow (30–50 days at room temperature) auto-catalytic reaction resulting in cleavage of the P—S bond to produce ethylmethylphosphonate (EMPA) and DIAESH (Diisopropylaminoethylmercaptan).
Caustic hydrolysis of Sarin also produces an alkyl phosphonate: specifically, isopropylmethylphosphonate (IMPA) and sodium fluoride.
As is seen, the phosphonate products of chemical hydrolysis are similar for these two agents: ethylmethylphosphonate (EMPA) is the byproduct of VX hydrolysis and isopropylmethylphosphonate (IMPA) is the byproduct of the hydrolysis of Sarin. In the past, biodegradation of these materials was accomplished using strategies similar to that for mustard—sequencing batch reactors. The reactors in a sequencing batch reactor system operate through a cycle of four discrete periods. The periods are fill, react, settle and draw. Waste is introduced into the reactor during fill. In such cases, the byproduct phosphonates were used as the sole phosphorus source for growth. Supplementary glucose was supplied simultaneously with the hydrolyzed agent. Although EMPA biodegradation proceeded well for the VX hydrolysate using sequencing batch reactors (DeFrank, J. J., I. J. Fry, J. P. Earley and R. L. Irvine, Biodegradation Studies with Water-Hydrolyzed Nerve Agent VX. Proceedings of the 20th Army Science Conference, p. 555–559 (1996), incorporated herein by reference in its entirety), poor results were exhibited for IMPA biodegradation when sludge sequencing batch reactors were employed for the Sarin hydrolysate (DeFrank, J. J., I. J. Fry, C. M. Frost and J. P. Earley, Sequencing Batch Reactor Biodegradation of Water-Hydrolyzed Sarin, Proceedings of the 1996 ERDEC Scientific Conference on Chemical and Biological Defense Research, p. 361–367 (1996), incorporated herein by reference in its entirety). Clearly, a better approach was needed to effectively degrade the IMPA needed for Sarin demilitarization purposes.
Prior work on alkylphosphonate biodegradation was reported by several investigators (Wanner, B. L, Phosphate-Regulated Genes for the Utilization of Phosphonates in Members of the Family Enterobacteriaceae, In: Phosphate in Microorganisms: Cellular and Molecular Biology, A. Torriani-Gorini, E. Yagil and S. Silver eds. ASM Press, Washington, D.C., pp. 215–221 (1994), incorporated herein by reference in its entirety). However, almost all of these reports focused on the mono-substituted phosphonates, such as methylphosphonate (MPA), ethylphosphonate (EPA), or α-aminoethylphosphonate (AEPN). The enzyme responsible for MPA biodegradation is C—P lyase. The reaction catalyzed by this enzyme is: 
C—P lyase is inhibited by low levels of phosphate (Daughton, C. G., A. M. Cook and M. Alexander, Bacterial Conversion of Alkylphosphonates to Natural Products via Carbon-Phosphorus Bond Cleavage, J. Agric. Food Chem. 27: 1375–1382 (1979)).
The biochemistry of MPA biodegradation has been well characterized in Enterobacter, Salmonella and E. coli (Wanner, B. L., Phosphate-Regulated Genes for the Utilization of Phosphonates in Members of the Family Enterobacteriacca, In: Phosphate in Microorganisms: Cellular and Molecular Biology, A. Torriani-Gorini, E. Yagil and S. Silver eds. ASM Press, Washington, D.C., pp. 215–221 (1994), incorporated herein by reference in its entirety). Several genes for MPA uptake and the biodegradation pathway were cloned and expressed in E. coli (Wanner, B. L. and J. A. Boline, Mapping and Molecular Cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli. J. Bacteriol. 172: 1186–1196 (1990), incorporated herein by reference in its entirety). IMPA biodegradation was reported in intracellular extracts of Pseudomonas testosteroni (Daughton, C. G., A. M. Cook and M. Alexander, Bacterial Conversion of Alkylphosphonates to Natural Products via Carbon-Phosphorus Bond Cleavage, J. Agric. Food Chem. 27: 1375–1382 (1979)). This organism could use several disubstituted alkylphosphonates as its sole phosphorus source but not as a sole carbon source.